Therapeutic platelets and methods

ABSTRACT

A dehydrated composition is provided that includes freeze-dried platelets. The platelets are loaded with trehalose which preserves biological properties during freeze-drying and rehydration. The trehalose loading is conducted at a temperature of from greater than about 25° C. to less than about 40° C., most preferably at 37° C., with the loading solution having trehalose in an amount from about 10 mM to about 50 mM. These freeze-dried platelets are substantially shelf-stable and are rehydratable so as to have a normal response to an agonist, for example, thrombin, with virtually all of the platelets participating in clot formation within about three minutes at 37° C.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with Government support under Grant No.HL67810-03, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

[0002] The present invention generally relates to the therapeutic usesof blood platelets, and more particularly to manipulations ormodifications of platelets, such as in preparing freeze-driedcompositions that can be rehydrated at the time of application and whichwhen rehydrated have a normal response to thrombin and other agonistswith respect to that of fresh platelets. The inventive compositions areuseful in applications such as transfusion therapy, as hemostasis aidsand for drug delivery.

BACKGROUND OF THE INVENTION

[0003] Blood transfusion centers are under considerable pressure toproduce platelet concentrates for transfusion. The enormous quest forplatelets necessitates storage of this blood component, since plateletsare important contributors to hemostasis. Platelets are generally ovalto spherical in shape and have a diameter of 2-4 μm. Today platelet richplasma concentrates are stored in bloodbags at 22° C.; however, theshelf life under these conditions is limited to five days. The rapidloss of platelet function during storage and risk of bacterialcontamination complicates distribution and availability of plateletconcentrates. Platelets tend to become activated at low temperatures.When activated they are substantially useless for an application such astransfusion therapy. Therefore the development of preservation methodsthat will increase platelet lifespan is desirable.

[0004] Several techniques for preservation of platelets have beendeveloped over the past few decades. Cryopreservation of platelets usingvarious agents, such as glycerol (Valeri et al., Blood, 43, 131-136,1974) or dimethyl sulfoxide, “DMSO” (Bock et al., Transfusion, 35,921-924, 1995), as the cryoprotectant have been done with some success.The best results have been obtained with DMSO. However, a considerablefraction of these cells are partly lysed after thawing and have theshape of a balloon. These balloon cells are not responsive to variousagonists, so that overall responsiveness of frozen thawed platelets tovarious agonists is reduced to less than 35% compared with freshplatelets. The shelflife of cryopreserved DMSO platelets at −80° C. isreported to be one year, but requires extensive washing and processingto remove cryoprotective agents, and even then the final product has asevere reduction in ability to form a clot.

[0005] Attempts to dry platelets by lyophilization have been describedwith paraformaldehyde fixed platelets (Read et al., Proc. Natl. Acad.Sci. USA, 92, 397-401, 1995). U.S. Pat. No. 5,902,608, issued May 11,1999, inventors Read et al. describe and claim a surgical aid comprisinga substrate on which fixed, dried blood platelets are carried. Thesedried blood platelets are fixed by contacting the platelets to afixative such as formaldehyde, paraformaldehyde, gutaraldehyde, orpermanganate. Proper functioning of lyophilized platelets that have beenfixed by such fixative agents in hemostasis is questionable.

[0006] Spargo et al., U.S. Pat. No. 5,736,313, issued Apr. 7, 1998, havedescribed a method in which platelets are loaded overnight with anagent, preferably glucose, and subsequently lyophilized. The plateletsare preincubated in a preincubation buffer and then are loaded withcarbohydrate, preferably glucose, having a concentration in the range ofabout 100 mM to about 1.5 M. The incubation is taught to be conducted atabout 10° C. to about 37° C., most preferably about 25° C.

[0007] U.S. Pat. No. 5,827,741, Beattie et al., issued Oct. 27, 1998,discloses cryoprotectants for human platelets, such as dimethylsulfoxideand trehalose. The platelets may be suspended, for example, in asolution containing a cryoprotectant at a temperature of about 22° C.and then cooled to below 15° C. This incorporates some cryoprotectantinto the cells.

[0008] Trehalose is a disaccharide found at high concentrations in awide variety of organisms that are capable of surviving almost completedehydration (Crowe et al., Anhydrobiosis. Annu. Rev. Physiol., 54,579-599, 1992). Trehalose has been shown to stabilize certain cellsduring freezing and drying (Leslie et al., Biochim. Biophys. Acta, 1192,7-13, 1994; Beattie et al., Diabetes, 46, 519-523, 1997).

[0009] Other workers have sought to load platelets with trehalosethrough use of electroporation before drying under vacuum. However,electroporation is very damaging to the cell membranes and is believedto activate the platelets. Activated platelets have dubious clinicalvalue.

[0010] Platelets have also been suggested for drug delivery applicationsin the treatment of various diseases, as is discussed by U.S. Pat. No.5,759,542, issued Jun. 2, 1998, inventor Gurewich. This patent disclosesthe preparation of a complex formed from a fusion drug including anA-chain of a urokinase-type plasminogen activator that is bound to anouter membrane of a platelet.

[0011] Accordingly, a need exists for the effective and efficientpreservation of platelets such that they maintain, or preserve, theirbiological properties, particularly their response to platelet agonistssuch as thrombin, and which can be practiced on a large, commerciallyfeasible scale. Further, it would also be useful to expand the types ofpresent vehicles that are useful for encapsulating drugs and used fordrug delivery to targeted sites.

SUMMARY OF THE INVENTION

[0012] In one aspect of the present invention, a dehydrated compositionis provided comprising freeze-dried platelets that are effectivelyloaded with trehalose to preserve biological properties duringfreeze-drying and rehydration. These platelets are rehydratable so as tohave a normal response to at least one agonist, such as thrombin. Forexample, substantially all freeze-dried platelets of the invention whenrehydrated and mixed with thrombin (1 U/ml) form a clot within threeminutes at 37° C. The dehydrated composition can include one or moreother agents, such as antibiotics, antifungals, growth factors, or thelike, depending upon the desired therapeutic application.

[0013] In another aspect of the invention, a hemostasis aid is providedwhere the above-described freeze-dried platelets are carried on or by abiocompatible surface. A further component of the hemostasis aid may bea therapeutic agent, such as an antibiotic, an antifungal, or a growthfactor. The biocompatible surface may be a bandage or a thrombicsurface, such as freeze-dried collagen. Such a hemostasis aid can berehydrated just before the time of application, such as by hydrating thesurface on or by which the platelets are carried, or, in case of anemergency, the dry hemostasis treatment aid could be applied directly tothe wound or burn and hydrated in situ.

[0014] Methods of making and using inventive embodiments are alsodescribed. One such method is a process of preparing a dehydratedcomposition comprising providing a source of platelets, effectivelyloading the platelets with trehalose to preserve biological properties,cooling the trehalose loaded platelets to below their freezing point,and lyophilizing the cooled platelets. The trehalose loading includesincubating the platelets at a temperature from greater than about 25° C.to less than about 40° C. with a trehalose solution having up to about50 mM trehalose therein. The process of using such a dehydratedcomposition further may comprise rehydrating the platelets. Therehydration preferably includes a prehydration step wherein thefreeze-dried platelets are exposed to warm, moisture saturated air for atime sufficient to bring the water content of the freeze-dried plateletsto between about 35 weight percent to about 50 weight percent.

[0015] In yet another aspect of the invention, a drug deliverycomposition is provided comprising platelets having a homogeneouslydistributed concentration of a therapeutic agent therein. The drugdelivery composition is particularly useful for targeting theencapsulated drug to platelet-mediated sites.

[0016] Practice of the invention permits the manipulation ormodification of platelets while maintaining, or preserving, biologicalproperties, such as a response to thrombin. Further, use of the methodto preserve platelets can be practiced on a large, commercially feasiblescale, and avoids platelet activation. The inventive freeze-driedplatelets, and hemostasis aids including the freeze-dried platelets, aresubstantially shelf stable at ambient temperatures when packaged inmoisture barrier materials.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In the drawings:

[0018]FIG. 1 graphically illustrates the loading efficiency of trehaloseplotted versus incubation temperature of human platelets;

[0019]FIG. 2 graphically illustrates the percentage of trehalose-loadedhuman platelets following incubation as a function of incubation time;

[0020]FIG. 3 graphically illustrates the internal trehaloseconcentration of human platelets versus external trehalose concentrationas a function of time at a constant temperature of 37° C.;

[0021]FIG. 4 graphically illustrates the loading efficiency of trehaloseinto human platelets as a function of extemal-trehalose concentration;

[0022]FIG. 5 graphically illustrates the recovery of plateletembodiments after lyophilization and direct rehydration with variousconcentrations of trehalose in the drying buffer, and in a combinationof 30 mM trehalose and one percent HSA in the drying buffer;

[0023]FIG. 6 graphically illustrates the uptake of FITC dextran versusthe external concentration compared with that of the marker, LYCH (withan incubation time of four hours);

[0024]FIG. 7 graphically illustrates the effect of prehydration onoptical density of platelets;

[0025]FIG. 8 illustrates the response of 500 μl platelets solution (witha platelet concentration of 0.5×10⁸ cells/ml) that was transferred toaggregation vials, thrombin added (1 U/ml) to each sample, and thesamples stirred for three minutes at 37° C., where panel (A) are theprior art platelets and panel (B) are the inventive platelets; and,

[0026]FIG. 9 graphically illustrates clot formation where the absorbancefalls sharply upon addition of thrombin (1 U/ml) and the plateletconcentration drops from 250×10⁶ platelets/mil to below 2×10⁶platelets/ml after three minutes for the inventive platelets.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Compositions and embodiments of the invention include plateletsthat have been manipulated (e.g. by freeze-drying) or modified (e.g.loaded with drugs), and that are useful for therapeutic applications,particularly for platelet transfusion therapy, as surgical or hemostasisaids, such as wound dressings, bandages, and as sutures, and asdrug-delivery vehicles. As has been known, human platelets have a phasetransition between 12° C. and 20° C. We have found that platelets have asecond phase transition between 30° C. and 37° C. Our discovery of thissecond phase transition temperature range suggests the possible use ofplatelets as vehicles for drug delivery because we can load plateletswith various useful therapeutic agents without causing abnormalitiesthat interfere with normal platelet responses due to changes, such as inthe platelet outer membranes.

[0028] For example, platelets may be loaded with anti-thrombic drugs,such as tissue plasminogen activator (TPA) so that the platelets willcollect at the site of a thrombus, as in an heart attack, and releasethe “clot busting” drug or drugs that are encapsulated and have beentargeted by the platelets. Antibiotics can also be encapsulated by theplatelets, since lipopolysaccharides produced by bacteria attractplatelets. Antibiotic loaded platelets will bring the selectedantibiotics to the site of inflammation. Other drugs that can be loadedinclude anti-mitotic agents and anti-angiogenic agents. Since plateletscirculate in newly formed vessels associated with tumors, they coulddeliver anti-mitotic drugs in a localized fashion, and likely plateletscirculating in the neovasculature of tumors can deposit anti-angiogenicdrugs so as to block the blood supply to tumors. Thus, platelets loadedwith a selected drug in accordance with this invention can be preparedand used for therapeutic applications. The drug-loaded platelets areparticularly contemplated for blood-borne drug delivery, such as wherethe selected drug is targeted to a site of platelet-mediated formingthrombi or vascular injury. The so-loaded platelets have a normalresponse to at least one agonist, particularly to thrombin. Suchplatelets can be loaded additionally with trehalose, if preservation byfreeze-drying is intended.

[0029] The key component for compositions and apparatus of theinvention, when preservation will be by freeze-drying, is anoligosaccharide, preferably trehalose, because we have found thatplatelets which are effectively loaded with trehalose preservebiological properties during freeze-drying (and rehydration). Thispreservation of biological properties, such as the normal clottingresponse in combination with thrombin, is necessary so that theplatelets following preservation can be successfully used in a varietyof therapeutic applications.

[0030] Normal hemostasis is a sequence of interactions in which bloodplatelets contribute, beginning with adhesion of platelets to an injuredvessel wall. The platelets form an aggregate that acceleratescoagulation. A complex, termed the glycoprotein (GP) 1b-IX-V complex, isinvolved in platelet activation by providing a binding site on theplatelet surface for the potent agonist, α-thrombin. α-thrombin is aserine protease that is released from damaged tissue. Thus, it isimportant that the manipulations and modifications in accordance withthis invention do not activate the platelets. Further, it is normallypreferred that the platelets be in a resting state. Otherwise, theplatelets will activate.

[0031] Although for most contemplated therapeutic applications theclotting response to thrombin is key, the inventive freeze-driedplatelets after rehydration will also respond to other agonists besidesthrombin. These include collagen, ristocetin, and ADP (adenosinediphosphate), all of which are normal platelet agonists. These otheragonists typically pertain to specific receptors on the platelet'ssurface.

[0032] Broadly, the preparation of preserved platelets in accordancewith the invention comprises the steps of providing a source ofplatelets, loading the platelets with a protective oligosaccharide at atemperature above about 25° C. and less than about 40° C., cooling theloaded platelets to below −32° C., and lyophilizing the platelets.

[0033] In order to provide a source of platelets suitable for theinventive preservation process, the platelets are preferably isolatedfrom whole blood. Thus, platelets used in this invention preferably havehad other blood components (erythrocytes and leukocytes) removed priorto freeze-drying. The removal of other blood components may be byprocedures well known to the art, which typically involve a centrifugestep.

[0034] The amount of the preferred trehalose loaded inside the inventiveplatelets is from about 10 mM to about 50 mM, and is achieved byincubating the platelets to preserve biological properties duringfreeze-drying with a trehalose solution that has up to about 50 mMtrehalose therein. Higher concentrations of trehalose during incubationare not preferred, as will be more fully explained later. The effectiveloading of trehalose is also accomplished by means of using an elevatedtemperature of from greater than about 25° C. to less than about 40° C.,more preferably from about 30° C. toeless than about 40° C., mostpreferably about 37° C. This is due to the discovery of the second phasetransition for platelets. As can be seen by FIG. 1, the trehaloseloading efficiency begins a steep slope increase at incubationtemperatures. above about 25° C. up to about 40° C. The trehaloseconcentration in the exterior solution (that is, the loading buffer) andthe temperature during incubation together lead to a trehalose uptakethat seems to occur primarily throughfluid phase endocytosis (that is,pinocytosis). Pinocytosed vesicles lyse over time, which results in ahomogeneous distribution of trehalose in the platelets, does notactivate the platelets, and can be applied for large scale production.FIG. 2 illustrates the trehalose loading efficiency as a function ofincubation time.

[0035] As may be gathered from various of the figures, in preparingparticularly preferred embodiments, platelets may be loaded withtrehalose by incubation at 37° C. for about four hours. The trehaloseconcentration in the loading buffer is preferably 35 mM, which resultsin an intracellular trehalose concentration of around 20 mM, but in anyevent is most preferably not greater than about 50 mM trehalose. Attrehalose concentrations below about 50 mM, platelets have a normalmorphological appearance.

[0036] Human platelets have a phase transition between 12° C. and 20° C.We found relatively poor loading when the platelets were chilled throughthe phase transition. Thus, in practicing the method described by U.S.Pat. No. 5,827,741, of which some of us are coinventors, only arelatively modest amount of trehalose may be loaded into platelets.

[0037] In this application, we have further investigated the phasetransition in platelets and have found a second phase transition between30° C. and 37° C. We believe that the excellent loading we obtain atabout 37° C. is in some way related to this second phase transition.Without being limited by theory, we also believe that pinocytosis isinvolved, but it may be that the second phase transition itselfstimulates the pinocytosis at high temperatures. It may be that otheroligosaccharides when loaded in this second phase transition in amountsanalogous to trehalose could have similar effects.

[0038] In any case, it is fortuitous that the loading can be done atelevated temperatures in view of the fact that chilling plateletsslowly—a requirement for using the first, or lower, phase transitionbetween 20° C. and 12° C. to introduce trehalose—is well known toactivate them (Tablin et al., J. Cell. Physiol., 168, 305-313, 1996).Our relatively high temperature loading, regardless of the mechanism, isthus unexpectedly advantageous both by providing increased loading aswell as surprisingly, obviating the activation problem.

[0039] Turning to FIG. 6, one sees that we have loaded other, largermolecules into the platelets. In FIG. 6 an illustrative large molecule(FITC dextran) was loaded into the platelets. This illustrates that awide variety of water-soluble, therapeutic agents can be loaded into theplatelets by utilizing the second phase transition, as we have shown maybe done with trehalose and with FITC dextran, while still maintainingcharacteristic platelet surface receptors and avoiding plateletactivation.

[0040] We have achieved loading efficiencies by practicing the inventionwith values as high as 61% after four hours incubation. The plateau isnot yet reached after four hours. The high loading efficiency oftrehalose is a strong indication that. the trehalose is homogeneouslydistributed rather than located in pinocytosed vesicles, and we expectsimilar results for loading other therapeutic agents. A loadingefficiency of 61% in an external concentration of 25 mM corresponds to acytosolic concentration of 15 mM. If trehalose was only located inendosomes of 0.1 micrometer, the vesiculation number would be more than1000. It is unlikely that such a high number of vesicles would bepresent in platelets next to the other platelet organelles. We thereforebelieve that the pinocytosed vesicles lyse in the cytoplasm. Thisresults in a homogeneous distribution of trehalose rather thanpunctuated loading in small vesicles. It is also possible that thetrehalose is crossing the membrane due to the phase transition between30° C. and 37° C.

[0041] We have found that the endocytotic uptake route is blocked atsugar concentrations above 0.1 M. Consequently, we prefer not to usesugar concentrations higher than about 50 mM in the loading buffer,because at some point above this value we have found swelling andmorphological changes of the platelets. Thus, we have found thatplatelets become swollen after four hours incubation at 37° C. in 75 mMtrehalose. Further, at concentrations higher than 50 mM the internaltrehalose concentration begins to decrease. By contrast to the presentinvention, the platelet method taught by Spargo et al., U.S. Pat. No.5,736,313, loads with carbohydrate in the range beginning at about 100mM and going up to 1.5 M. As noted, we find a high concentration ofloading buffer, at least with trehalose, to lead to swelling andmorphological changes.

[0042] The effective loading of platelets with trehalose is preferablyconducted by incubating for at least about two hours, preferably for atleast about four hours. After this loading, then the platelets arecooled to below their freezing point and lyophilized.

[0043] Before freezing, the platelets should be placed into a restingstate. If not in the resting state, platelets would likely activate. Inorder to place the platelets in a resting state, a variety of suitableagents, such as calcium channel blockers, may be used. For example,solutions of adenine, adenosine or iloprost are suitable for thispurpose. Another suitable agent is PGE1. It is important that theplatelets are not swollen and are completely in the resting state priorto drying. The more they are activated, the more they will be damagedduring freeze-drying.

[0044] After the platelets have been effectively loaded with trehaloseand are in a resting state, then the loading buffer is removed and theplatelets are contacted with a drying buffer. Drying of platelets aftertrehalose loading may be carried out by suspending the platelets in asolution containing a suitable water replacing molecule (or dryingbuffer), such as albumin. If albumin is used, it should be from the samespecies as the platelets. The drying buffer should also includetrehalose, preferably in amounts up to about 100 mM. The trehalose inthe drying buffer assists in spatially separating the platelet as wellas stabilizing the platelet membranes on the exterior. The drying bufferpreferably also includes a bulking agent (to further separate theplatelets). As already mentioned, albumin may serve as a bulking agent,but other polymers may be used with the same effect. Suitable otherpolymers, for example, are water-soluble polymers such as HES anddextran.

[0045] The trehalose loaded platelets in drying buffer are then cooledto a temperature below about −32° C. A cooling, that is, freezing, rateis preferably between −30° C. and −1° C./min. and more preferablybetween about −2° C./min to −5° C./min.

[0046] The lyophilization step is preferably conducted at a temperaturebelow about −32° C., for example conducted at about −40° C., and dryingmay be continued until about 95 weight percent of water has been removedfrom the platelets. During the initial stages of lyophilization, thepressure is preferably at about 1×10⁻⁶ torr. As the samples dry, thetemperature can be raised to be warmer than −32° C. Based upon the bulkof the sample, the temperature and the pressure it can be empericallydetermined what the most efficient temperature values should be in orderto maximize the evaporative water loss. Freeze-dried compositions of theinvention preferably have less than about 5 weight percent water.

[0047] The freeze-dried platelets may be used by themselves, dissolvedin a physiologically acceptable solution, or may be a component of abiologically compatible (biocompatible) structure or matrix, whichprovides a surface on or by which the freeze-dried platelets arecarried. The freeze-dried platelets can be, for example, applied as acoating to or impregnated in a wide variety of known and usefulmaterials suitable as biocompatible structures for therapeuticapplications. The earlier mentioned U.S. Pat. No. 5,902,608, forexample, discusses a number of materials useful for surgical aid, wounddressings, bandages, sutures, prosthetic devices, and the like. Sutures,for example, can be monofilament or braided, can be biodegradable ornonbiodegradable, and can be made of materials such as nylon, silk,polyester, cotton, catgut, homopolymers, and copolymers of glycolide andlactide, etc. Polymeric materials can also be cast as a thin film,sterilized, and packaged for use as a wound dressing. Bandages may bemade of any suitable substrate material, such as woven or nonwovencotton or other fabric suitable for application to or over a wound, mayoptionally include a backing material, and may optionally include one ormore adhesive regions on the face surface thereof for securing thebandage over the wound.

[0048] The freeze-dried platelets, whether by themselves, as a componentof a vial-compatible structure ornatrix, and optionally including otherdry or freeze-dried components, may be packaged so as to preventrehydration until desired. The packaging may be any of the varioussuitable packagings for therapeutic purposes, such as made from foil,metallized plastic materials, and moisture barrier plastics (e.g.highdensity polyethylene or plastic films that have been created withmaterials such as SiOx), cooling the trehalose loaded platelets to belowtheir freezing point, and lyophilizing the cooled platelets. Thetrehalose loading includes incubating the platelets at a temperaturefrom greater than about 25° C. to less than about 40° C. with atrehalose solution having up to about 50 mM trehalose therein. Theprocess of using such a dehydrated composition comprises rehydrating theplatelets. The rehydration preferably includes a prehydration stepsufficient to bring the water content of the freeze-dried platelets tobetween 35 weight percent to about 50 weight percent.

[0049] When reconstitution is desired, prehydration of the freeze-driedplatelets in moisture saturated air followed by rehydration ispreferred. Use of prehydration yields cells with a much more denseappearance and with no balloon cells being present. Prehydrated,previously lyophilized platelets of the invention resemble freshplatelets. This is illustrated, for example, by FIG. 7. As can be seen,the previously freeze-dried platelets can berestored to a condition thatlooks like fresh platelets.

[0050] Before the prehydration step, it is desirable to have diluted theplatelets in the drying buffer to prevent aggregation during theprehydration and rehydration. At concentrations below about 3×10⁸cells/ml, the ultimate recovery is about 70% with no visible aggregates.Prehydration is preferably conducted in moisture saturated air, mostpreferably is conducted at about 37° C. for about one hour to aboutthree hours. The preferred prehydration step brings the water content ofthe freeze-dried platelets to between about 35 weight percent to about50 weight percent.

[0051] The prehydrated platelets may then be fully rehydrated.Rehydration may be with any aqueous based solutions, depending upon theintended application. In one preferred rehydration, we have used plasma,which has resulted in about 90% recovery.

[0052] Since it is frequently desirable to dilute the platelets toprevent aggregation when the freeze-dried platelets are once againhydrated, it may then be desired or necessary for particular clinicalapplications to concentrate the platelets. Concentration can be by anyconventional means, such as by centrifugation. In general, a rehydratedplatelet composition will preferably have 10⁶ to 10¹¹ platelets per ml,more preferably 10⁸ to 10¹⁰ platelets per ml.

[0053] By contrast with the previous attempts at freeze dryingplatelets, we show here that with a very simple loading, freeze-dryingand rehydration protocol one obtains platelets that are morphologicallyintact after rehydration, and have an identical response to thrombin asdo fresh platelets. Moreover, the concentration of thrombin to give thisresponse is a physiological concentration—1 U/ml.

[0054] For example, FIG. 8, panel (A), illustrates the clot formationfor fresh platelets and in panel (B) for platelets that have beenpreserved and then rehydrated in accordance with this invention. Thecell counts that were determined after three minutes exposure tothrombin were zero for both the fresh platelets and the previouslyfreeze-dried platelets of the invention.

[0055]FIG. 9 graphically illustrates clotting as measured with anaggregometer. With this instrument one can measure the change intransmittance when a clot is formed. The initial platelet concentrationwas 250×10⁶ platelets/ml, and then thrombin (1 U/ml) was added and theclot formation was monitored with the aggregometer. The absorbance fellsharply and the cell count dropped to below 2×10⁶ platelets/ml afterthree minutes, which was comparable to the results when the test was runwith fresh platelets as a control.

[0056] Although platelets for use in this invention preferably have hadother blood components removed before freeze-drying, compositions andapparatuses of the invention may also include a variety of additionaltherapeutic agents. For example, particularly for embodimentscontemplated in hemostasis applications, antifungal and antibacterialagents are usefully included with the platelets, such as being admixedwith the platelets. Embodiments can also include admixtures orcompositions including freeze-dried collagen, which provides athrombogenic surface for the platelets. Other components that canprovide a freeze-dried extracellular matrix can be used, for example,components composed of proteoglycan. Yet other therapeutic agents thatmay be included in inventive embodiments are growth factors. When theembodiments include such other components, or admixtures, they arepreferably in dry form, and most preferably are also freeze-dried. Wealso contemplate therapeutic uses of the composition where additionaltherapeutic agents may be incorporated into or admixed with theplatelets in hydrated form. The platelets, as earlier mentioned, canalso be prepared as to encapsulate drugs in drug delivery applications.If trehalose is also loaded into the platelet interiors, then suchdrugencapsulated platelets may be freeze-dried as has been earlierdescribed.

[0057] The platelets should be selected of the mammalian species forwhich treatment is intended (e.g. human, equine, canine, feline, orendangered species), most preferably human.

[0058] The injuries to be treated by applying hemostasis aids with theplatelets include abrasions, incisions, bums, and may be woundsoccurring during surgery of organs or of skin tissue. The platelets ofthe invention may be applied or delivered to the location of such injuryor wound by any suitable means. For example, application of inventiveembodiments to bums can encourage the development of scabs, theformation of chemotactic gradients, of matrices for inducing bloodvessel growth, and eventually for skin cells to move across and fill inthe burn.

[0059] For transfusion therapy, inventive compositions may bereconstituted (rehydrated) as pharmaceutical formulations andadministered to human patients by intravenous injection. Suchpharmaceutical formulations may include any aqueous carrier suitable forrehydrating the platelets (e.g., sterile, physiological saline solution,including buffers and other therapeutically active agents that may beincluded in the reconstituted formulation). For drug delivery, theinventive compositions will typically be administered into the bloodstream, such as by i.v.

[0060] Aspects of the invention will now be illustrated by the followingexamples, which are not intended to limit the invention. Abbreviationsused in the examples, and elsewhere, are as follows.

[0061] DMSO=dimethylsulfoxide

[0062] ADP=adenosine diphosphate

[0063] PGE1=prostaglandin E1

[0064] HES=hydroxy ethyl starch

[0065] EGTA=ethylene glycolbis(2aminoethyl ether)N,N,N′,N′, tetraaceticacid

[0066] TES=Ntris (hydroxymethyl) methyl2aminoethanesulfonic acid

[0067] HEPES=N(2hydroxyl ethyl) piperarineN′(2ethanesulfonic acid)

[0068] PBS=phosphate buffered saline

[0069] HSA=human serum albumin

EXPERIMENTAL Example 1

[0070] Washing of Platelets. Platelet concentrations were obtained fromthe Sacramento blood center or from volunteers in our laboratory.Platelet rich plasma was centrifuged for 8minutes at 320×g to removeerythrocytes and leukocytes. The supernatant was pelleted and washed twotimes (480×g for 22 minutes, 480×g for 15 minutes) in buffer A (100 mMNaCl, 10 mM KCl, 10mM EGTA, 10 mM imidazole, pH 6.8). Platelet countswere obtained on a Coulter counter T890 (Coulter, Inc., Miami, Fla.).

[0071] Loading of Lucifer Yellow CH into Platelets. A fluorescent dye,lucifer yellow CH (LYCH), was used as a marker for penetration of themembrane by a solute. Washed platelets in a concentration of 1-2×10⁹platelets/ml were incubated at various temperatures in the presence of1-20 mg/ml LYCH. Incubation temperatures and incubation times werechosen as indicated. After incubation the platelets suspensions werespun down for 20× at 14,000 RPM (table centrifuge), resuspended inbuffer A, spun down for 20 s in buffer A and resuspended. Plateletcounts were obtained on a Coulter counter and the samples were pelleted(centrifugation for 45 s at 14,000 RPM, table centrifuge). The pelletwas lysed in 0.1% Tritonbuffer (10 mM TES, 50 mM KCl, pH 6.8 ). Thefluorescence of the lysate was measured on a PerkinElmer LS5spectrofluorimeter with excitation at 428 nm (SW 10 nm) and emission at530 nm (SW 10 nm). Uptake was calculated for each sample as nanograms ofLYCH per cell using a standard curve of LYCH in lysate buffer. Standardcurves of LYCH, were found to be linear up to 2000 nm ml⁻¹.

[0072] Visualization of cellassociated lucifer yellow. LYCH loadedplatelets were viewed on a fluorescence microscope (Zeiss) employing afluorescein filter set for fluorescence microscopy. Platelets werestudied either directly after incubation or after fixation with 1%paraformaldehyde in buffer. Fixed cells were settled on polyLlysinecoated cover slides and mounted in glycerol.

[0073] Loading of Platelets with Trehalose. Washed platelets in aconcentration of 1-2 10⁹ platelets/ml were incubated at varioustemperatures in the presence of 1-20 mg/ml trehalose. Incubationtemperatures were chosen from 4° C. to 37° C. Incubation times werevaried from 0.5 to 4 hours. After incubation the platelet solutions werewashed in buffer A two times (by centrifugation at 14,000 RPM for 20 sin a table centrifuge). Platelet counts were obtained on a coultercounter. Platelets were pelleted (45 S at 14,000 RPM) and sugars wereextracted from the pellet using 80% methanol. The samples were heatedfor 30 minutes at 80° C. The methanol was evaporated with nitrogen, andthe samples were kept dry and redissolved in H₂O) prior to analysis. Theamount of trehalose in the platelets was quantified using the anthronereaction (Umbreit et al., Mamometric and Biochemical Techniques, 5^(th)Edition, 1972). Samples were redissolved in 3 ml H₂O and 6 ml anthronereagents (2 g anthrone dissolved in 1 l sulfuric acid). After vortexmixing, the samples were placed in a boiling water bath for 3 minutes.Then the samples were cooled on ice and the absorbance was measured at620 nm on a Perkin Elmer spectrophotometer. The amount of plateletassociated trehalose was determined using a standard curve of trehalose.Standard curves of trehalose were found to be linear from 6 to 300 μgtrehalose per test tube.

[0074] Quantification of Trehalose and LYCH Concentration. Uptake wascalculated for each sample as micrograms of trehalose or LYCH perplatelet. The internal trehalose concentration was calculated assuming aplatelet radius of 1.2 μm and by assunmng that 50% of the plateletvolume is taken up by the cytosol (rest is membranes). The loadingefficiency was determined from the cytosolic trehalose or LYCHconcentration and the concentration in the loading buffer.

[0075]FIG. 1 shows the effect of temperature on the loading efficiencyof trehalose into human platelets after a 4 hour incubation period with50 mM external trehalose. The effect of the temperature on the trehaloseuptake showed a similar trend as the LYCH uptake. The trehalose uptakeis relatively low at temperatures of 22° C. and below (below 5%), but at37° C. the loading efficiency of trehalose is 35% after 4 hours.

[0076] When the time course of trehalose uptake is studied at 37° C., abiphasic curve can be seen (FIG. 2). The trehalose uptake is initiallyslow (2.8×10⁻¹¹ mol m² s from 0 to 2 hours), but after 2 hours a rapidlinear uptake of 3.3×10⁻¹⁰ mol/m² s can be observed. The loadingefficiency increases up to 61% after an incubation period of 4 hours.This high loading efficiency is a strong indication that the trehaloseis homogeneously distributed in the platelets rather than located inpinocytosed vesicles.

[0077] The uptake of trehalose as a function of the external trehaloseconcentration is shown in FIG. 3. The uptake of trehalose is linear inthe range from 0 to 30 mM external trehalose. The highest internaltrehalose concentration is obtained with 50 mM external trehalose. Athigher concentrations than 50 mM the internal trehalose concentrationdecreases again. Even when the loading buffer at these high trehaloseconcentrations is corrected for isotonicity by adjusting the saltconcentration, the loading efficiency remains low. Platelets becomeswollen after 4 hours incubation in 75 mM trehalose.

[0078] The stability of the platelets during a 4 hours incubation periodwas studied using microscopy and flow cytometric analysis. Nomorphological changes were observed after 4 hours incubation ofplatelets at 37° C. in the presence of 25 mM external trehalose. Flowcytometric analysis of the platelets showed that the platelet populationis very stable during 4 hours incubation. No signs of microvesicleformation could be observed after 4 hours incubation, as can be judgedby the stable relative proportion of microvesicle gated cells (less than3%). The formation of microvesicles is usually considered as the firstsign of platelet activation (Owners et al., Trans. Med. Rev., 8 , 27-44,1994). Characteristic antigens of platelet activation include:glycoprotein 53 (GP53 , a lysosomal membrane marker), PECAM-1(plateletendothelial cell adhesion molecule-1, an alpha granuleconstituent), and Pselectin (an alpha granule membrane protein).

Example 2

[0079] Washing Platelets. Platelets were obtained from volunteers in ourlaboratory. Platelet rich plasma was centrifuged for 8 minutes at 320×gto remove erythrocytes and leukocytes. The supernatant was pelleted andwashed two times (480×g for 22 minutes, 480×g for 15 minutes) in bufferA (100 mM NaCl, 10 mM KCl, 10 mM EGTA, 10 mM imidazole, 10 μg/ml PGE1,pH 6.8). Platelet counts were obtained on a Coulter counter T890(Coulter, Inc., Miami, Fla.).

[0080] Loading Platelets with Trehalose. Platelets were loaded withtrehalose as described in Example 1. Washed platelets in a concentrationof 1-2×10⁹ platelets/ml were incubated at 37° C. in buffer A with 35 mMtrehalose added. Incubation times were typically 4 hours. The sampleswere gently stirred for 1 minute every hour. After incubation theplatelet solutions were pelleted (25 sec in a microfuge) and resuspendedin drying buffer (9.5 mM HEPES, 142.5 mM NaCl, 4.8 mM KCl, 1 mM MgCl₂,30 mM Trehalose, 1% Human Serum Albumin, 10 μg/ml PGE1). In theaggregation studies no PGE1 was added in the drying buffer. Trehalosewas obtainedfrom Pfahnstiehl. A 30% human serum albumin was obtainedfrom Sigma.

[0081] Freezing and Drying. Typically 0.5 ml platelet suspensions weretransferred in 2 ml Nunc cryogenic vials and frozen in a Cryomedcontrolled freezing device. Vials were frozen from 22° C. to −40° C.with freezing rates between −30 and −1° C./min and more often between −5and −2° C./min. The frozen solutions were transferred to a −80° C.freezer and kept there for at least half an hour. Subsequently thefrozen platelet suspensions were transferred in vacuum flasks that wereattached to a Virtis lyophilizer. Immediately after the flasks werehooked up to the lyophilizer, they were placed in liquid nitrogen tokeep the samples frozen until the vacuum returned to 20×10⁻⁶ Torr, afterwhich the samples were allowed to warm to the sublimation temperature.The condenser temperature was −45° C. Under these conditions, sampletemperature during primary drying is about −40° C., as measured with athermocouple in the sample. It is important to maintain the sample belowT_(g) for the excipient during primary drying (−32° C. for trehalose).

[0082] Rehydration. Vials with. originally 0.5 ml platelet suspensionwere rehydrated in 1 ml PBS buffer/water (1/1). PBS buffer was composedof 9.4 mM Na2HPO₄, 0.6 mM KH₂PO₄, 100 mM NaCl). In a few experimentsPGE1 was added to the rehydration buffer in a condition of 10 μg/ml orrehydration was performed in plasma/water (1/1).

[0083] Prehydration. Platelet lyophilisates were prehydrated in a closedbox with moisture saturated air at 37° C. Prehydration times werebetween 0 and 3 hours.

[0084] Recovery. The numerical recovery of lypophilized and(p)rehydrated platelets was determined by comparing the cell count witha Coulter count T890 (Coulter, Inc., Miami, Florida) before drying andafter rehydration. The morphology of the rehydrated platelets wasstudied using a light microscope. For this purpose platelets were fixedin 2% paraformaldehyde or gutaraldehyde and allowed to settle onpolyLlysine coated coverslides for at least 45 minutes. After this thecoverslides were mounted and inspected under the microscope. The Opticaldensity of freeze-dried and rehydrated platelets was determined bymeasuring the absorbance of a platelet suspension of 1.0×10⁸ cells/ml at550 nm on a Perkin Elmer absorbance spectrophotometer.

[0085] Aggregation studies. Dried platelets were rehydrated (after 2hour prehydration) with 2 aliquots of platelet free plasma (plasma wascentrifuged for 5 minutes at 3800×g) diluted with water in 1/1 ratio.Half ml aliquots of this platelet suspension were transferred toaggregation cuvettes with a magnetic stirrer. The response of theplatelets to thrombin was tested by adding thrombin (1 U/ml) to theplatelet suspension at 37° C. under stirring conditions. After 3 minutesthrombin treated platelet suspensions were inspected for clots and cellcounts were done on a Coulter Counter T890.

[0086] Direct rehydration tends toward cell lysis and prehydration leadsto aggregation when the cell concentration is 10⁹ cells/ml in the dryingbuffer. We found also that recovery of prehydrated and rehydratedplatelets depends on the cell concentration in the dryingbuffer. Therecovery drops to very low values if the cell concentration is higherthan 3×10⁸ cells/ml. At concentrations below 3×10⁸ cells/ml, therecovery is around 70%, and no aggregates were visible. Prehydrationresulted in denser cells and the absence of balloon cells.

[0087] Longer prehydration times than 90 minutes did not further improvethe cellular density, but slightly activated the platelets. The watercontent of the pellet increases with increasing prehydration time, andpreferably is between about 35% and 50% at the moment of rehydration. Athigher water contents than 50% water droplets become visible in thelyophilisate (which means that the platelets are in a very hypertonicsolution).

[0088] As described by Example 1, platelets were loaded with trehaloseby incubation at 37° C. for 4 hours in buffer A with 35 mM trehalose,which yielded platelets with intracellular trehalose concentration of15-25 mM. After incubation, the platelets were transferred to dryingbuffer with 30 mM trehalose and 1% HSA as the main excipients.

[0089] The directly rehydrated platelets had a high numerical recoveryof 85%, but a considerable fraction (25-50%) of the cells was partlylysed and had the shape of a balloon. Directly rehydrated platelets wereoverall less dense when compared with fresh platelets.

[0090] The numerical recovery of platelets that were prehydrated inmoisture saturated air was only 25% when the platelet concentration was1×10⁹ cells/ml in the drying buffer. This low recovery was due toaggregates that were formed during the prehydration period. But thecells that were not aggregated were more dense than the directlyrehydrated platelets and resembled that of fresh platelets.

[0091] Since it appears desirable to dilute the platelets to preventaggregation during the prehydration step, it may be necessary forclinical applications to concentrate the platelets followingrehydration. We therefore also tested the stability of the rehydratedplatelets with respect to centrifugation and found that the directlyrehydrated platelets had 50% recovery after centrifugation, while theprehydrated ones had 75% recovery following centrifugation. Thus, weconclude that the inventive platelets can be concentrated without illeffect.

Example 3

[0092] We view trehalose as the main lyoprotectant in the drying buffer.However, other components in the drying buffer, such as albumin, canimprove the recovery. In the absence of external trehalose in dryingbuffer, the numerical recovery is only 35%. With 30 mM trehalose in thedrying buffer the recovery is around 65%. A combination of 30 mMtrehalose and 1% albumin gave a numerical recovery of 85%.

Example 4

[0093] Typically 0.5 ml platelet suspensions were transferred in 2 mlNunc cryogenic vials and frozen in a Cryomed controlled freezing device.Vials were frozen from 22° C. to −40° C. with freezing rates between−30° C./min and −1° C./min and more often between −5° C. and −2° C./min.The frozen solutions were transferred to a −80° C. freezer and keptthere for at least half an hour. Subsequently the frozen plateletsuspensions were transferred in vacuum flasks that were attached to aVirtus lyophilizer Immediately after the flsks were hooked up to thelyophilizer, they were placed in liquid nitrogen to keep the samplesfrozen until the vacuum returned to 20×10⁻⁶ Torr, after which thesamples were allowed to warm to the sublimation temperature. Thecondensortemperature was −45° C. Under these conditions, sampletemperature during primary drying is about −40° C., as measured with athermocouple in the sample. In is important to maintain the sample belowT_(g), for the excipient during primary drying (−32° C. for trehalose).Only minor differences in recovery were found as a function of thefreezing rate. The optimal freezing rate was found to be between 2° C.and 5° C./minute.

Example 5

[0094] Response of freeze-dried platelets to thrombin (1 U/ml) wascompared with that of fresh platelets. The platelet concentration was0.5×10⁸ cells/ml in both samples. 500 μl platelets solution wastransferred into aggregation vials. Thrombin was added to the samplesand the samples were stirred for 3 minutes at 37° C. The cell countsthat were determined after 3 minutes were 0 for both the fresh and thefreeze-dried platelets. The response to thrombin was determined by acleavage in glycoprotein 1b-(GP1b). This was detected by usingmonoclonal antibodies and flow cytometry. Thus, the pattern seen afteraddition of thrombin was a reduced amount of GP1b on the plateletsurface.

[0095] The response of lyophilized, prehydrated, and rehydratedplatelets (Examples 1 and 2) to thrombin (1 U/ml) was found to beidentical compared with that of fresh platelets. In both fresh andrehydrated platelets a clot was formed within 3 minutes at 37° C. Theseclots are illustrated by FIG. 8, panels (A) and (B). When cell countswere done with the Coulter counter, we found no cells present,indicating that all platelets participated in forming the clotillustrated in panel (1).

Example 6

[0096] Reactions with of the agonists were studied. Platelet suspensionsof the, inventive platelets were prepared with 50×10⁶ platelets/ml.Different agonists were then added and subsequently counted with aCoulter counter to determine the percentage of platelets involved in thevisually observable clot formation. The cell count was between 0 and2×10⁶ platelets/ml:

[0097] after 5 minutes with 2 mg/ml collagen

[0098] after 5 minutes with 20 μM ADP

[0099] after 5 minutes with 1.5 mg/ml ristocetin

[0100] This means that the percentage of platelets that are involved inclot formation is between 95-100% for all the agonists tested. Theagonist concentrations that were used are all physiological. In allcases the percentage of clotted platelets was the same as fresh controlplatelets.

[0101] It is to be understood that while the invention has beendescribed above in conjunction with preferred specific embodiments, thedescription and examples are intended to illustrate and not limit thescope of the invention, which is defined by the scope of the appendedclaims.

1. A dehydrated composition, useful for mammalian therapy, comprising: substantially shelf-stable freeze-dried platelets selected from the mammalian species for which therapy is intended, the platelets being effectively loaded with trehalose to preserve biological properties during freeze-drying and rehydration, wherein the platelets are rehydratable so as to have a normal response to at least one agonist.
 2. The dehydrated composition as in claim 1 wherein the amount of trehalose loaded inside the freeze-dried blood platelets is from about 10 mM to about 50 mM.
 3. The dehydrated composition as in claim 1 wherein the normal response to at least one agonist is a response to thrombin in a physiological concentration.
 4. The dehydrated composition as in claim 1 wherein the preserved biological properties are mediated via characteristic platelet surface receptors.
 5. The dehydrated composition as in claim 1 wherein the at least one agonist is selected from the group consisting of thrombin, collagen, ristocetin, and ADP.
 6. The dehydrated composition as in claim 1 wherein the composition is substantially shelf stable at ambient temperatures.
 7. The dehydrated composition as in claim 1 wherein the effective loading includes incubating platelets at a temperature from greater than about 25° C. to less than about 40° C. so as to uptake external trehalose via fluid phase endocytosis.
 8. The dehydrated composition as in claim 1 wherein the platelets are human platelets.
 9. The dehydrated composition as in claim 1 wherein the freeze-dried platelets before freeze-drying are characterized by a homogenous distribution of trehalose therein of about 20 mM.
 10. The dehydrated composition as in claim 1 wherein moisture is in an amount not greater than about 5 weight percent.
 11. The dehydrated composition as in claim 1 further including a therapeutic agent selected from the group consisting of an antibiotic, an antifungal, a growth factor, and mixtures thereof.
 12. A therapeutic composition, comprising: platelets having a homogeneously distributed concentration of a therapeutic agent therein, the platelets determinable to have a normal response to thrombin.
 13. The therapeutic composition as in claim 12 wherein the determinable normal response to thrombin is clot formation within about three minutes at 37° C.
 14. The therapebutic composition as in claim 12 wherein the therapeutic agent includes an anti-thrombic agent, an antibiotic, an anti-mitotic agent or an anti-angiogenic agent.
 15. A hemostasis aid, comprising: substantially shelf-stable freeze-dried platelets selected from the mammalian species for which therapy is intended, the platelets being effectively loaded with trehalose to preserve biological properties during freeze-drying and rehydration, wherein the platelets are rehydratable so as to have a normal response to at least one agonist; and, a biocompatible matrix on which the platelets are carried.
 16. The hemostasis aid as in claim 15 wherein the platelets are coated on or impregnated in the matrix.
 17. The hemostasis aid as in claim 15 wherein the matrix is a woven or non-woven bandage, wound dressing, or suture.
 18. A process of preparing a dehydrated composition, useful for therapy to a mammal, comprising: providing platelets selected from the mammalian species for which therapy is intended, the platelets being effectively loaded with an oligosaccharide to preserve biological properties, wherein the loading includes incubating the platelets at a temperature from greater than about 25° C. to less than about 40° C. with an oligosaccharide solution, the solution having up to about 50 mM oligosaccharide therein, the incubating sufficient to load oligosaccharide inside the platelets in an amount from about 10 mM to about 50 mM; cooling the loaded platelets to below their freezing point; and, lyophilizing the cooled platelets.
 19. The process as in claim 18 wherein the platelets are human platelets.
 20. The process as in claim 18 wherein the incubating temperature is about 37° C.
 21. The process as in claim 18 wherein the incubating is for at least about two hours.
 22. The process as in claim 18 wherein the incubating is for at least about four hours.
 23. The process as in claim 18 wherein the cells are human platelets, the incubating is between about 30° C. and about 37° C., the solution has trehalose in an amount between about 20 mM and 50 mM, and the incubation is for at least about four hours.
 24. The process as in claim 23 wherein the cooling is at a rate of about 2° C. to 5° C. per minute and is conducted in a drying buffer.
 25. The process as in claim 18 wherein the lyophilizing is conducted at a temperature below about −32° C. and removes about 95 weight percent of water.
 26. A therapeutic process of using a dehydrated composition, comprising: providing freeze-dried platelets selected from a mammalian species for which therapy is intended, the platelets being effectively loaded with trehalose to preserve biological properties; and, applying the freeze-dried platelets to a wound or burn of the selected mammalian species.
 27. The process as in claim 26 wherein the freeze-dried platelets are carried on a biologically compatible matrix.
 28. The process as in claim 26 wherein the freeze-dried platelets are rehydrated prior to or upon application to the wound or burn.
 29. The process as in claim 26 wherein the freeze-dried platelets are prehydrated in moisture saturated air before application.
 30. The process as in claim 29 wherein the prehydrated, freeze-dried platelets are rehydrated following prehydration.
 31. The process as in claim 29 wherein the prehydration is conducted at about 37° C. for between about one hour to about three hours.
 32. The process as in claim 29 wherein the prehydration is sufficient to bring the water content of the freeze-dried platelets to between about 35 weight percent to about 50 weight percent. 